For the diagnosis and prophylaxis of diabetes mellitus, the importance of periodic monitoring of blood glucose levels has been increasingly emphasized. Nowadays, strip-type biosensors designed for hand-held reading devices, which are usually based on colorimetry or electrochemistry, allow individuals to readily monitor glucose levels in the blood.
The electrochemistry applied to many commercially available biosensors for the analysis of blood glucose levels is explained by the following Reaction Formula I, featuring the use of an electron transfer mediator (M).Glucose+GOx-FAD→gluconic acid+GOx-FADH2 GOx-FADH2+Mox→GOx-FAD+Mred  [Reaction Formula I]
wherein, GOx represents glucose oxidase; GOx-FAD and GOx-FADH2 respectively represent an oxidized and a reduced state of glucose-associated FAD (flavin adenine dinucleotide), a cofactor required for the catalyst of glucose oxidase; and Mox and Mred denote an oxidized and a reduced state of an electron transfer mediator, respectively.
Examples of the electron transfer mediator for use in electrochemical biosensors include: ferrocene and derivatives thereof; quinines and derivatives thereof; transition metal, containing organic or inorganic compounds, such as hexamine ruthenium, osmium-containing polymers, potassium ferricyanide, etc.; organic conducting salts; and viologen.
In the biosensor, blood glucose levels are measured on the basis of the following principle.
Glucose in the blood is oxidized to gluconic acid by the catalysis of glucose oxidase with the concomitant reduction of the cofactor FAD to FADH2. Then, the reduced cofactor FADH2 transfers electrons to the mediator, so that FADH2 returns to its oxidized state; that is, FAD and the mediator are reduced. The reduced mediator is diffused to the surface of the electrodes. The series of reaction cycles is driven by the anodic potential applied at the working electrode, and redox current proportional to the level of glucose is measured. Over biosensors based on colorimetry, the electrochemical biosensors (that is, based on electrochemistry) have the advantages of not being influenced by oxygen and allowing the use of samples, even if cloudy, without pretreatment thereof.
Sample introduction channels of conventional biosensors are largely divided into “i” type and “—” type.
An “i” type sample introduction channel is typically structured to have an opening at an end of a straight sampling passage so as to induce a capillary phenomenon. However, such an “i” type sample introduction channel is problematic in that, depending on the viscosity thereof, the sample introduced through the straight sampling passage may overflow or may not be able to reach the opening located at the end of the straight channel, incurring non-uniformity in sample amount.
A “—” type sample introduction channel is typically structured to have a passage extending from a lateral side of the sensor to another lateral side. Because it is located at a lateral side and has a “—” type structure, the sample introduction channel is inconvenient for a user to introduce a sample therethrough. In addition, turbulence or eddies form upon sample introduction, making it impossible to introduce a sample in a constant amount.
In order to overcome these problems, suggested is an assay device in which the flow in a reagent chamber with the port narrowing just after the entry of a sample and in a hydrophobic zone is controlled by a time gate, thereby preventing turbulence or eddies (U.S. Pat. No. 6,156,270). However, this assay device is not suitable for use with samples on the microliter scale because a reaction-inducible reagent is mixed with a sample before a detection region and the hydrophobic zone is wide and has a wave form. That is, this assay device is not applicable to the case where the mixing of reagents and a sample and detection reactions must be implemented within a narrow cell.
In a conventional biosensor, a sample introduction channel is constructed in a double-sided tape or a film over a working electrode, an auxiliary electrode or a reference electrode, and is lined with an enzyme reaction solution. The sample introduction channel, however, is disadvantageous in that the sample introduction channel is greatly affected by a cut-out pattern of the double-sided tape or laminated film so that it is non-uniformly lined with the enzyme reaction solution, or the amount of the sample that is introduced is not constant, incurring errors in measurement.
Leading to the present invention, intensive and thorough research into the introduction of a constant amount of a sample into an electrochemical biosensor for accurate analysis resulted in the finding that an insulator with a structural pattern that is aligned with a capillary tube pattern of a medium plate can control turbulence or eddies effectively, thus introducing a sample uniformly into a biosensor and increasing analysis accuracy.